Key Challenges for In Vitro Testing of Tobacco Products for Regulatory Applications: Recommendations for the In Vitro Mouse Lymphoma Assay.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across traditional tobacco and various tobacco and nicotine next-generation products (NGPs), including Heated Tobacco Products (HTPs) and Electronic Nicotine Delivery Systems (ENDS). This report was developed by a working group composed of attendees of the seventh IIVS workshop, 'Approaches and recommendations for conducting the mouse lymphoma gene mutation assay (MLA) and introduction to in vitro disease models', which was held virtually on 21-23 June 2022. This publication provides a background overview of the MLA, and includes the description of assay conduct and data interpretation, key challenges and recommended best practices for evaluating tobacco and nicotine products, with a focus on the evaluation of NGPs, and a summary of how the assay has been used to evaluate and compare tobacco and nicotine products.

Development of a framework for risk assessment of dietary carcinogens.

Although there are a number of guidance documents and frameworks for evaluation of carcinogenicity, none of the current methods fully reflects the state of the science. Common limitations include the absence of dose-response assessment and not considering the impact of differing exposure patterns (e.g., intermittent, high peaks vs. lower, continuous exposures). To address these issues, we have developed a framework for risk assessment of dietary carcinogens. This framework includes an enhanced approach for weight of evidence (WOE) evaluation for genetic toxicology data, with a focus on evaluating studies based on the most recent testing guidance to determine whether a chemical is a mutagen. Included alongside our framework is a discussion of resources for evaluating tissue dose and the temporal pattern of internal dose, taking into account the chemical's toxicokinetics. The framework then integrates the mode of action (MOA) and associated dose metric category with the exposure data to identify the appropriate approach(es) to low-dose extrapolation and level of concern associated with the exposure scenario. This framework provides risk managers with additional flexibility in risk management and risk communication options, beyond the binary choice of linear low-dose extrapolation vs. application of uncertainty factors.

PBPK modeling to evaluate maximum tolerated doses: A case study with 3-chloroallyl alcohol.

Introduction: A physiologically based pharmacokinetic (PBPK) model for 3-chloroallyl alcohol (3-CAA) was developed and used to evaluate the design of assays for the in vivo genotoxicity of 3-CAA. Methods: Model development was supported by read across from a published PBPK model for ethanol. Read across was motivated by the expectation that 3-CAA, which like ethanol is a primary alcohol, is metabolized largely by hepatic alcohol dehydrogenases. The PBPK model was used to evaluate how two metrics of tissue dosimetry, maximum blood concentration (Cmax; mg/L) and area under the curve (AUC; mg-hr/L) vary with dose of 3-CAA and with dose route (oral gavage, drinking water). Results: The model predicted that oral gavage results in a 6-fold higher Cmax than the same dose administered in drinking water, but in similar AUCs. Predicted Cmax provided the best correlation with severe toxicity (e.g., lethality) from 3-CAA, consistent with the production of a reactive metabolite. Therefore, drinking water administration can achieve higher sustained concentration without severe toxicity in vivo. Discussion: This evaluation is significant because cytotoxicity is a potential confounder of mutagenicity testing. The PBPK model can be used to ensure that studies meet OECD and USEPA test guidelines and that the highest dose used is not associated with severe toxicity. In addition, PBPK modeling provides assurance of target tissue (e.g., bone marrow) exposure even in the absence of laboratory data, by defining the relationship between applied dose and target tissue dose based on accepted principles of pharmacokinetics, relevant physiology and biochemistry of the dosed animals, and chemical-specific information.

Key Challenges and Recommendations for In Vitro Testing of Tobacco Products for Regulatory Applications: Consideration of Test Materials and Exposure Parameters.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific and technical approaches for conducting in vitro assays, to assess potential toxicity within and across tobacco and various next generation nicotine and tobacco products (NGPs), including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDS). The third workshop (24-26 February 2020) summarised the key challenges and made recommendations concerning appropriate methods of test article generation and cell exposure from combustible cigarettes, HTPs and ENDS. Expert speakers provided their research, perspectives and recommendations for the three basic types of tobacco-related test articles: i) pad-collected material (PCM); ii) gas vapour phase (GVP); and iii) whole smoke/aerosol. These three types of samples can be tested individually, or the PCM and GVP can be combined. Whole smoke/aerosol can be bubbled through media or applied directly to cells at the air-liquid interface. Summaries of the speaker presentations and the recommendations developed by the workgroup are presented. Following discussion, the workshop concluded the following: that there needs to be greater standardisation in aerosol generation and collection processes; that methods for testing the NGPs need to be developed and/or optimised, since simply mirroring cigarette smoke testing approaches may be insufficient; that understanding and quantitating the applied dose is fundamental to the interpretation of data and conclusions from each study; and that whole smoke/aerosol approaches must be contextualised with regard to key information, including appropriate experimental controls, environmental conditioning, analytical monitoring, verification and performance criteria.

Key challenges for in vitro testing of tobacco products for regulatory applications: Recommendations for dosimetry.

The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to develop recommendations for optimal scientific and technical approaches for conducting in vitro assays to assess potential toxicity within and across tobacco and various next-generation products (NGPs) including heated tobacco products (HTPs) and electronic nicotine delivery systems (ENDSs). This publication was developed by a working group of the workshop members in conjunction with the sixth workshop in that series entitled "Dosimetry for conducting in vitro evaluations" and focuses on aerosol dosimetry for aerosol exposure to combustible cigarettes, HTP, and ENDS aerosolized tobacco products and summarizes the key challenges as well as documenting areas for future research.

A comparison of cigarette smoke test matrices and their responsiveness in the mouse lymphoma assay: A case study.

No cigarette smoke test matrix is without limitation, due to the complexity of the starting aerosol and phase to phase dynamics. It is impossible to capture all chemicals at the same level of efficiency, therefore, any test matrix will inadvertently or by design fractionate the test aerosol. This case study examines how four different test matrices derived from cigarette smoke can be directly compared. The test matrices assessed were as follows, total particulate matter (TPM), gas vapour phase (GVP), a combination of TPM + GVP and whole aerosol (WA). Here we use an example assay, the mouse lymphoma assay (MLA) to demonstrate that data generated across four cigarette smoke test matrices can be compared. The results show that all test matrices were able to induce positive mutational events, but with clear differences in the biological activity (both potency and toxicity) between them. TPM was deemed the most potent test article and by extension, the particulate phase is interpreted as the main driver of genotoxic induced responses in the MLA. However, the results highlight that the vapour phase is also active. MLA appeared responsive to WA, with potentially lower potency, compared to TPM approaches. However, this observation is caveated in that the WA approaches used for comparison were made on a newly developed experimental method using dose calculations. The TPM + GVP matrix had comparable activity to TPM alone, but interestingly induced a greater number of mutational events at comparable relative total growth (RTG) and TPM-equivalent doses when compared to other test matrices. In conclusion, this case study highlights the importance of understanding test matrices in response to the biological assay being assessed and we note that not all test matrices are equal.

A critical review and meta-analysis of epidemiology studies of occupationally exposed styrene workers evaluated for chromosomal aberration incidence.

Chromosome aberrations in the peripheral blood lymphocytes of styrene exposed workers have been suggested as a potential early marker for cancer risk. We performed a critical review and abstracted data from all studies using current chromosome aberration scoring criteria and providing at least a mean and standard deviation or standard error for the exposed and comparison groups. Using these data, we conducted a meta-analysis of occupational styrene exposed workers and incidence of chromosome aberrations. Our meta-analysis used the standardized mean difference as the summary statistic since all studies assess the same outcome but use different comparison populations. The primary meta-analysis of the 20 comparisons of 505 styrene exposed workers to 532 comparison workers found a meta-mean difference of 0.361 (95 % CI -0.084 to 0.807, random effects model), but there was substantial lack of consistency across studies (I2 of 90.11, p-value <0.001, fixed effect model). Studies with higher styrene exposures had lower mean standard differences compared to studies with lower styrene exposures. While studies of styrene workers overall had a slight increase in chromosomal aberrations relative to comparison groups, the lack of consistency across studies and the absence of an exposure response and other limitations of the reviewed studies including inadequate exposure assessment, small numbers of participants per study, and poorly matched exposed and comparison workers, we find insufficient evidence to support a conclusion that styrene exposure increases chromosome aberration frequencies in styrene workers.

Revisiting the bacterial mutagenicity assays: Report by a workgroup of the International Workshops on Genotoxicity Testing (IWGT).

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing "proficiency compounds" to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.

Detection of Loss of Heterozygosity in Tk-Deficient Mutants from L5178Y Tk+/--3.7.2C Mouse Lymphoma Cells.

The mouse lymphoma assay (MLA), a forward mutation assay using the Tk+/--3.7.2C clone of the L5178Y mouse lymphoma cell line and the Thymidine kinase (Tk) gene, has been widely used as an in vitro genetic toxicity assay for more than four decades. The MLA can evaluate the ability of mutagens to induce a wide range of genetic events including both gene mutations and chromosomal mutations and has been recommended as one component of several genotoxicity test batteries. Tk-deficient mutants often exhibit chromosomal abnormalities involving the distal end of chromosome 11 where the Tk gene is located, in mice, and the type of chromosome alteration can be analyzed using a loss of heterozygosity (LOH) approach. LOH has been considered an important event in human tumorigenesis and can result from any of the following several mechanisms: large deletions, mitotic recombination, and chromosome loss. In this chapter, the authors describe the procedures for the detection of LOH in the Tk mutants from the MLA, and apply LOH analysis for understanding the types of genetic damage that is induced by individual chemicals.

Recommended criteria for the evaluation of bacterial mutagenicity data (Ames test).

A committee was constituted within the International Workshop on Genetic Toxicology Testing (IWGT) to evaluate the current criteria for a valid Ames test and to provide recommendations for interpretation of test results. Currently, determination of a positive vs. a negative result is made by applying various data evaluation procedures for comparing dosed plates with the concurrent solvent control plates. These evaluation procedures include a requirement for a specific fold increase (2- or 3-fold, specific to the bacterial strain), formal statistical procedures, or subjective (expert judgment) evaluation. After extensive discussion, the workgroup was not able to reach consensus recommendations in favor of any of these procedures. There was a consensus that combining additional evaluation criteria to the comparison between dosed plates and the concurrent solvent control plates improves test interpretation. The workgroup recommended using these additional criteria because the induction of mutations is a continuum of responses and there is no biological relevance to a strict dividing line between a positive (mutagenic) and not-positive (nonmutagenic) response. The most useful additional criteria identified were a concentration-response relationship and consideration of a possible increase above the concurrent control in the context of the laboratory's historical solvent control values for the particular tester strain. The workgroup also emphasized the need for additional testing to resolve weak or inconclusive responses, usually with altered experimental conditions chosen based on the initial results. Use of these multiple criteria allowed the workgroup to reach consensus on definitions of "clear positive" and "clear negative" responses which would not require a repeat test for clarification. The workgroup also reached consensus on recommendations to compare the responses of concurrent positive and negative controls to historical control distributions for assay acceptability, and the use of control charts to determine the validity of the individual test.

Demonstrating laboratory proficiency in bacterial mutagenicity assays for regulatory submission.

The bacterial reverse mutation test is a mainstay for evaluation of mutagenicity predicting the carcinogenic potential of a test substance and is recommended by regulatory agencies across the globe. The popularity of the test is due, in part, to the relatively low cost, rapid results and small amount of test material required compared to most other toxicological tests as well as the near universal acceptance of the toxicological significance of a clear positive or negative result. Most laboratories follow the Organization for Economic Cooperation and Development Test Guideline 471 (TG471) or national guidelines based on TG471. Regulatory agencies in most countries are obligated to consider results from tests which meet the recommendations laid out in TG471. Nonetheless, laboratories unfamiliar with the test sometimes have trouble generating reliable, reproducible results. TG471 is a test guideline, not a detailed test protocol. A group of experts from regulatory agencies and laboratories which use the assay has assembled here a set of recommendations which if followed, will allow an inexperienced laboratory to acquire proficiency in assay conduct. These include recommendations for how to create a cell bank for the 5 Salmonella typhimurium/Escherichia coli strains and develop a laboratory protocol to reliably culture each strain to ensure each culture has the characteristics which allow adequate sensitivity for detection of mutagens using the test as described in TG471. By testing compounds on the provided lists of positive and negative test substances, the laboratory will have surmounted many of the problems commonly encountered during routine testing of unknown chemicals and will have gained the experience necessary to prepare the detailed protocol needed for performing the test under Good Laboratory Procedures and the laboratory will have generated the historical positive and negative control databases which are needed for test reports which adhere to TG471.

Horizon scanning for novel and emerging in vitro mammalian cell mutagenicity test systems.

The induction of gene mutation within a DNA sequence can result in an adverse impact, altering or preventing gene function. Therefore, in vitro evaluation of mutagenicity is an essential component of the toxicological screening process. A variety of mutagen screening tools are routinely used in genetic toxicology, which are based on selected reporter genes. These assays are however typically labour intensive and impractical for high throughput screening. Considering this, the IWGT (International Workshops on Genotoxicity Testing) sub-group on Novel & Emerging in vitro Mammalian Cell Mutagenicity Test Systems undertook a literature search to identify new approaches for mutation detection. This review therefore focused on identifying new approaches for mutation detection that have the potential for use as a future genotoxicity screening tool. A comprehensive literature review identified genome-wide loss-of-function screening tools, next generation sequencing (NGS) mutation characterisation and fluorescence-based mutation detection methods as having significant promise as an emerging in vitro mammalian cell mutagenicity test system. Each of the technologies considered was assessed for its capacity to report on a wide array of heritable mutagenic changes, necessary to cover the full spectrum of genetic events imparted by substances with a broad range of modes of action. Of the technologies evaluated, NGS techniques exhibited the greatest advantages for use in a genotoxicity testing setting. However, it is important to note that the emerging techniques identified could not facilitate routine mutagenicity testing in their current format and require substantial additional optimisation and tailoring before they could be utilised as an in vitro mammalian cell mutagenicity test system. Additionally, new mammalian cell mutation test systems must be able to accurately and reliably detect and quantify rare events; hence any new system would require careful validation. Nevertheless, with further development emerging technologies such as NGS could become important in establishing more predictive and high-throughput regulatory hazard screening tools of the future.

In Vitro Mouse Lymphoma Cell (L5178Y Tk+/- -3.7.2.C) Forward Mutation Assay.

The in vitro mouse lymphoma cell assay (MLA) is one of the most widely practiced assays in genetic toxicology. MLA detects forward mutations at the thymidine kinase (Tk) locus of the L5178Y (Tk+/- -3.7.2C) cell line derived from a mouse thymic lymphoma. This assay is capable of detecting a wide range of genetic events including point mutations, deletions and multilocus, chromosomal rearrangements, mitotic recombination and nondisjunction. There are two equally accepted versions of the assay, one using soft agar cloning and the second method using liquid media cloning in 96-microwell plates. There are two morphologically distinct types of mutant colonies recovered in the MLA; small and large colony mutants. The induction of small colony mutants is associated with chemicals inducing gross chromosomal aberrations, whereas the induction of large mutant colonies is generally associated with chemicals inducing point mutations. The source and karyotype of the cell line as well as the culture conditions are important variables that could influence the assay performance. The assay when performed according to the standards recommended by the International Workshops on Genotoxicity Testing (IWGT) and the Organization of Economic Cooperation and Development Test Guideline 490 is capable of providing valuable genotoxicity hazard information as part of the overall safety assessment process of various classes of test substances.

The food contaminant acetamide is not an in vivo clastogen, aneugen, or mutagen in rodent hematopoietic tissue.

Acetamide (CAS 60-35-5) is classified by IARC as a Group 2B, possible human carcinogen, based on the induction of hepatocellular carcinomas in rats following chronic exposure to high doses. Recently, acetamide was found to be present in a variety of human foods, warranting further investigation. The regulatory body JECFA has previously noted conflicting reports on acetamide's ability to induce micronuclei (MN) in mice in vivo. To better understand the potential in vivo genotoxicity of acetamide, we performed acute MN studies in rats and mice, and a subchronic study in rats, the target species for liver cancer. In the acute exposure, animals were gavaged with water vehicle control, 250, 1000, or 2000 mg/kg acetamide, or the positive control (1 mg/kg mitomycin C). In the subchronic assay, bone marrow of rats gavaged at 1000 mg/kg/day (limit dose) for 28 days was evaluated. Both acute and subchronic exposures showed no change in the ratio of polychromatic to total erythrocytes (P/E) at any dose, nor was there any increase in the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Potential mutagenicity of acetamide was evaluated in male rats gavaged with vehicle control or 1500 mg/kg/day acetamide using the in vivoPig-a gene mutation assay. There was no increase in mutant red blood cells or reticulocytes in acetamide-treated animals. In both acute and sub-chronic studies, elevated blood plasma acetamide in treated animals provided evidence of systemic exposure. We conclude based on this study that acetamide is not clastogenic, aneugenic, or mutagenic in vivo in rodent hematopoietic tissue warranting a formal regulatory re-evaluation.

Critical review of styrene genotoxicity focused on the mutagenicity/clastogenicity literature and using current organization of economic cooperation and development guidance.

Styrene is an important high production volume chemical used to manufacture polymeric products. In 2018, International Agency for Research on Cancer classified styrene as probably carcinogenic to humans; National Toxicology Program lists styrene as reasonably anticipated to be a human carcinogen. The genotoxicity literature for styrene and its primary metabolite, styrene 7,8-oxide (SO), begins in the 1970s. Organization of Economic Cooperation and Development (OECD) recently updated most genotoxicity test guidelines, making substantial new recommendations for assay conduct and data evaluation for the standard mutagenicity/clastogenicity assays. Thus, a critical review of the in vitro and in vivo rodent mutagenicity/clastogenicity studies for styrene and SO, based on the latest OECD recommendations, is timely. This critical review considered whether a study was optimally designed, conducted, and interpreted and provides a critical assessment of the evidence for the mutagenicity/clastogenicity of styrene/SO. Information on the ability of styrene/SO to induce other types of genotoxicity endpoints is summarized but not critically reviewed. We conclude that when styrene is metabolized to SO, it can form DNA adducts, and positive in vitro mutagenicity/clastogenicity results can be obtained. SO is mutagenic in bacteria and the in vitro mouse lymphoma gene mutation assay. No rodent in vivo mutation studies were identified. SO is clastogenic in cultured mammalian cells. Although the in vitro assays gave positive responses, styrene/SO is not clastogenic/aneugenic in vivo in rodents. In addition to providing updated information for styrene, this review demonstrates the application of the new OECD guidelines for chemicals with large genetic toxicology databases where published results may or may not be reliable. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

A meta-analysis of epidemiologic studies of occupationally exposed styrene workers and micronuclei levels.

Previous reviews and meta-analyses have reached different conclusions on whether high styrene exposure increase micronucleus frequencies. The most recent meta-analysis reports an increase in these frequencies related to styrene. We update this meta-analysis of micronucleus frequencies with additional studies not previously included, eliminate double counting of study subjects appearing in more than one publication, considered levels of styrene exposures in the analysis, assess publication bias, and examine consistency of findings across studies. Our meta-analysis used the standardized mean difference as the summary statistic since all studies assess the same outcome but use different comparison populations. The primary meta-analysis of the 12 studies of 516 styrene exposed workers and 497 non-exposed comparisons produces a meta-mean difference of 1.19 (95% CI 0. 20-2.18, random effects model), but there was substantial heterogeneity across studies (I2 of 97.47, p-value <0.001, fixed effect model). We also observed that studies with higher styrene exposure had a higher mean standard difference compared with studies with lower styrene exposures. However, a longitudinal study did not find any association with styrene exposure and micronucleus frequencies. Given the lack of consistency across studies and the equivocal finding on exposure response, these data are insufficient to support a conclusion that an increase in micronucleus frequencies is due to styrene exposure.

Genotoxicity Assessment of Nanomaterials: Recommendations on Best Practices, Assays, and Methods.

Nanomaterials (NMs) present unique challenges in safety evaluation. An international working group, the Genetic Toxicology Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, has addressed issues related to the genotoxicity assessment of NMs. A critical review of published data has been followed by recommendations on methods alterations and best practices for the standard genotoxicity assays: bacterial reverse mutation (Ames); in vitro mammalian assays for mutations, chromosomal aberrations, micronucleus induction, or DNA strand breaks (comet); and in vivo assays for genetic damage (micronucleus, comet and transgenic mutation assays). The analysis found a great diversity of tests and systems used for in vitro assays; many did not meet criteria for a valid test, and/or did not use validated cells and methods in the Organization for Economic Co-operation and Development Test Guidelines, and so these results could not be interpreted. In vivo assays were less common but better performed. It was not possible to develop conclusions on test system agreement, NM activity, or mechanism of action. However, the limited responses observed for most NMs were consistent with indirect genotoxic effects, rather than direct interaction of NMs with DNA. We propose a revised genotoxicity test battery for NMs that includes in vitro mammalian cell mutagenicity and clastogenicity assessments; in vivo assessments would be added only if warranted by information on specific organ exposure or sequestration of NMs. The bacterial assays are generally uninformative for NMs due to limited particle uptake and possible lack of mechanistic relevance, and are thus omitted in our recommended test battery for NM assessment. Recommendations include NM characterization in the test medium, verification of uptake into target cells, and limited assay-specific methods alterations to avoid interference with uptake or endpoint analysis. These recommendations are summarized in a Roadmap guideline for testing.

Development of an adverse outcome pathway for chemically induced hepatocellular carcinoma: case study of AFB1, a human carcinogen with a mutagenic mode of action.

Adverse outcome pathways (AOPs) are frameworks starting with a molecular initiating event (MIE), followed by key events (KEs) linked by KE relationships (KERs), ultimately resulting in a specific adverse outcome. Relevant data for the pathway and each KE/KER are evaluated to assess biological plausibility, weight-of-evidence, and confidence. We aimed to describe an AOP relevant to chemicals directly inducing mutation in cancer critical gene(s), via the formation of chemical-specific pro-mutagenic DNA adduct(s), as an early critical step in tumor etiology. Such chemicals have mutagenic modes-of-action (MOA) for tumor induction. To assist with developing this AOP, Aflatoxin B1 (AFB1) was selected as a case study because it has a rich database and is considered to have a mutagenic MOA. AFB1 information was used to define specific KEs, KERs, and to inform development of a generic AOP for mutagen-induced hepatocellular carcinoma (HCC). In assessing the AFB1 information, it became clear that existing data are, in fact, not optimal and for some KEs/KERs, the definitive data are not available. In particular, while there is substantial information that AFB1 can induce mutations (based on a number of mutation assays), the definitive evidence - the ability to induce mutation in the cancer critical gene(s) in the tumor target tissue - is not available. Thus, it is necessary to consider the patterns of results in the weight-of-evidence for KEs and KERs. It was important to determine whether there was sufficient evidence that AFB1 can induce the necessary critical mutations early in the carcinogenic process, which was the case.

Comparative Genotoxicity of TEMPO and 3 of Its Derivatives in Mouse Lymphoma Cells.

TEMPO (2, 2, 6, 6-tetramethylphiperidine-1-oxyl) and its derivatives are stable free radical nitroxides widely used in the field of chemistry, biology, and pharmacology. TEMPO was previously found to be mutagenic and to induce micronuclei in mammalian cells. In this study, we investigated and quantified the genotoxicity of 4 structurally similar nitroxides, TEMPO and 3 of its derivatives (4-hydroxy-TEMPO, 4-oxo-TEMPO, and 4-methoxy-TEMPO), using the mouse lymphoma assay (MLA) and Comet assay in L5178Y Tk+/- cells. The results showed that all tested nitroxides were cytotoxic and mutagenic in the MLA, both in the presence and absence of S9, with metabolic activation significantly enhancing the cytotoxicity and/or mutagenicity. In addition, the 4 nitroxides caused DNA-strand breakage. The mutagenicity and DNA damaging dose-responses of the test articles were compared using the PROAST benchmark dose software package. The potency ranking of the 4 nitroxides for mutagenicity was different from the ranking of the DNA damaging effects. The mode of action analysis by a multi-endpoint DNA damage pathway assay classified all 4 nitroxides as clastogens. In addition, the majority of the induced Tk mutants showed loss of heterozygosity at the Tk and D11Mit42 loci (ie, chromosome damage <31 Mbp). These results suggest that TEMPO and its 3 derivatives are cytotoxic and mutagenic in mouse lymphoma cells through a mechanism that involves strand breakage and large alterations to DNA. The potency rankings indicate that the different TEMPO derivatives vary in their mutagenic and DNA damaging potential.

Analysis of negative historical control group data from the in vitro micronucleus assay using TK6 cells.

The recent revisions of the Organisation for Economic Co-operation and Development (OECD) genetic toxicology test guidelines emphasize the importance of historical negative controls both for data quality and interpretation. The goal of a HESI Genetic Toxicology Technical Committee (GTTC) workgroup was to collect data from participating laboratories and to conduct a statistical analysis to understand and publish the range of values that are normally seen in experienced laboratories using TK6 cells to conduct the in vitro micronucleus assay. Data from negative control samples from in vitro micronucleus assays using TK6 cells from 13 laboratories were collected using a standard collection form. Although in some cases statistically significant differences can be seen within laboratories for different test conditions, they were very small. The mean incidence of micronucleated cells/1000 cells ranged from 3.2/1000 to 13.8/1000. These almost four-fold differences in micronucleus levels cannot be explained by differences in scoring method, presence or absence of exogenous metabolic activation (S9), length of treatment, presence or absence of cytochalasin B or different solvents used as vehicles. The range of means from the four laboratories using flow cytometry methods (3.7-fold: 3.5-12.9 micronucleated cells/1000 cells) was similar to that from the nine laboratories using other scoring methods (4.3-fold: 3.2-13.8 micronucleated cells/1000 cells). No laboratory could be identified as an outlier or as showing unacceptably high variability. Quality Control (QC) methods applied to analyse the intra-laboratory variability showed that there was evidence of inter-experimental variability greater than would be expected by chance (i.e. over-dispersion). However, in general, this was low. This study demonstrates the value of QC methods in helping to analyse the reproducibility of results, building up a 'normal' range of values, and as an aid to identify variability within a laboratory in order to implement processes to maintain and improve uniformity.